The present invention concerns a method for the detection of cytosine methylations in DNA.
The levels of observation that have been well studied due to method developments in recent years in molecular biology include the genes themselves, as well as [transcription and] translation of these genes into RNA and the proteins arising therefrom. During the course of development of an individual, when a gene is turned on and how the activation and inhibition of certain genes in certain cells and tissues are controlled can be correlated with the extent and nature of the methylation of the genes or of the genome. Pathogenic states are also expressed by a modified methylation pattern of individual genes or of the genome.
5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, in genetic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. 5-Methylcytosine positions, however, cannot be identified by sequencing, since 5-methylcytosine has the same base-pairing behavior as cytosine. In addition, in the case of a PCR amplification, the epigenetic information, which is borne by 5-methylcytosines, is completely lost.
A relatively new method that has since been applied most frequently for investigating DNA for 5-methylcytosine is based on the specific reaction of bisulfite with cytosine, which is converted to uracil, which corresponds in its base-pairing behavior to thymidine, after a subsequent alkaline hydrolysis. In contrast, 5-methylcytosine is not modified under these conditions. Thus the original DNA is converted, such that methylcytosine, which originally cannot be distinguished from cytosine by means of its hybridization behavior, now can be detected by “standard” molecular biological techniques as the single remaining cytosine, for example, by amplification and hybridization or sequencing. All of these techniques are based on base pairing, which now is fully utilized. The prior art, which concerns sensitivity, is defined by a method that incorporates the DNA to be investigated in an agarose matrix, through which diffusion and renaturation of the DNA is prevented (bisulfite reacts only on single-stranded DNA) and all precipitation and purification steps are replaced by rapid dialysis (Olek A., et al., Nucl. Acids Res. 1996, 24, 5064-5066). Individual cells can be investigated with this method, which illustrates the potential of the method. Of course, previously, only individual regions of up to approximately 3000 base pairs in length have been investigated; a global investigation of cells for thousands of possible methylation analyses is not possible. Of course, this method also cannot reliably analyze very small fragments comprised of small sample quantities. These are lost despite the protection from diffusion through the matrix.
A review of the other known possibilities for detecting 5-methylcytosines can be derived from the following review article: Rein T., DePamphilis, M. L., Zorbas H., Nucleic Acids Res. 1998, 26, 2255.
The bisulfite technique has previously been applied only in research, with a few exceptions (e.g., Ze[s]chnigk M. et al., Eur. J Hum. Gen. 1997, 5, 94-8). However, short, specific pieces of a known gene are always amplified after a bisulfite treatment and either completely sequenced (Olek A. and Walter, J., Nat. Genet. 1997, 17, 275-276) or individual cytosine positions (Gonzalgo M. L. and Jones P. A., Nucl. Acids Res. 1997, 25, 2529-2531, WO Patent 95 00669) or an enzyme cleavage (Xiong Z. and Laird P. W., Nucl. Acids Res. 1997, 25, 2532-2534) are detected by a “primer extension reaction”. Also, detection by means of hybridizing has been described (Olek et al., WO 99/28498).
Other publications, which are concerned with the application of the bisulfite technique for the detection of methylation in individual genes are: Xiong, Z. and Laird, P. W. (1997), Nucl. Acids Res. 25, 2532; Gonzalgo, M. L. and Jones, P. A. (1997) Nucl. Acids Res. 25, 2529; Grigg S. and Clark S. (1994) Bioassays 16, 431; Zeschnigk, M. et al, (1997) Human Molecular Genetics 6, 387; Teil R. et al. (1994), Nucl. Acids Res. 22, 695; Martin V. et al. (1995), Gene. 157, 261; WO 97/46705, WO 95/15373 and WO 45560.
A review of the prior art in oligomer array production can be taken from a special publication of Nature Genetics that appeared in January 1999 (Nature Genetics Supplement, Volume 21, January 1999), the literature cited therein and U.S. Pat. No. 5,994,065 on methods for the production of solid carriers for target molecules such as oligonucleotides with reduced nonspecific background signal.
Probes with many fluorescent labels have been used for the scanning of an immobilized DNA array. Particularly suitable for fluorescent labels is the simple introduction of Cy3 and Cy5 dyes at the 5′-OH of the respective probe. The fluorescence of the hybridized probes is detected, for example, by means of a confocal microscope. The dyes Cy3 and Cy5, in addition to many others, are commercially available.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) is a very powerful development for the analysis of biomolecules (Karas M., and Hillenkamp F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. (1998) Anal. Chem. 60, 2299-2301). An analyte is embedded in a light-absorbing matrix. The matrix is evaporated by means of a short laser pulse and the analyte molecule is transported unfragmented into the gas phase. The ionization of the analyte is achieved by collisions with matrix molecules. An applied voltage accelerates the ions in a field-free flight tube. The ions are accelerated to a varying extent based on their different masses. Smaller ions reach the detector sooner than larger ions.
MALDI-TOF spectroscopy is excellently suitable for the analysis of peptides and proteins. The analysis of nucleic acids is somewhat more difficult (Gut, I. G. and Beck, S. (1995)), DNA and Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Molecular Biology: Current Innovations and Future Trends 1: 147-157.) For nucleic acids, the sensitivity is approximately 100 times poorer than for peptides and decreases overproportionally with increasing fragment size. For nucleic acids, which have a backbone with multiple negative charges, the ionization process through the matrix is essentially less efficient. In MALDI-TOF spectroscopy, the selection of the matrix plays a very important role. For the desorption of peptides, several very powerful matrices have been found, which produce a very fine crystallization. Several high-performing matrices have been found in the meantime for DNA, but the difference in sensitivity has not been reduced in this way. The difference in sensitivity can be reduced by modifying the DNA chemically in such a way that it is similar to a peptide.
Phosphorothioate nucleic acids, in which the usual phosphates of the backbone are substituted by thiophosphates, can be converted into a charge-neutral DNA by simple alkylation chemistry (Gut, I. G. and Beck, S. (1995), A procedure for selective DNA alkylation and detection by mass spectrometry. Nucleic Acids Res. 23: 1367-1373). The coupling of a “charge tag” to this modified DNA results in an increase in sensitivity by the same amount that is found for peptides. Another advantage of “charge tagging” is the increased stability of the analysis against impurities, which greatly interfere with the detection of unmodified substrates.
Genomic DNA is obtained by standard methods from DNA of cells, tissue or other test samples. This standard methodology is found in references such as Fritsch and Maniatis, eds., Molecular Cloning: A Laboratory Manual, 1989.
Urea improves the efficiency of the bisulfite treatment prior to sequencing of 5-methylcytosine in genomic DNA (Paulin, R. Grigg G W, Davey M W, Piper M. (1998), Nucleic Acids Res. 26: 5009-5010).